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Abstract

Silicon (Si) is a biologically important element for plants in the order Poales (Yamamoto et al., 2011; Kido et al., 2015). In rice, Si is mainly deposited in the motor cells and the cell walls of the leaf epidermis. However, the molecular basis of this overall process has not been elucidated. Thus, we propose a protocol for the histochemical staining of the silica body based on specific hydrogen bonding between silanol group and the carboxylate group of crystal violet lactone (Ichimura et al., 2008), as described by Isa et al. (2010), but with minor modifications. This modified protocol can be used for observing Si accumulation during rice development.

Rice (Oryza sativa L. cv. Nipponbare) plants were grown in a liquid medium supplemented with 1.5 mM SiO32- in a growth chamber at 28 °C under a 15/9 h light/dark cycle (light at 150 μ mol-2 s-1). This protocol is performed with the fifth leaf blade at foliar age 5.2 (Kido et al., 2015). The foliar age “m.n” is defined as those in which the mth leaf is fully expanded and the (m+1)th leaf is under development with a length of n/10 of the fully expanded-length

Prepare the fixative by dissolving 0.4 g of paraformaldehyde powder in 10 ml of 20 mM sodium cacodylate buffer (pH 7.4). Heat this solution in a fume hood on the hotplate/stirrer to approximately 70 °C until the solution clears completely, and allow it to cool. The fixative mixture should be prepared immediately before use.

Cut the rice leaf blade into pieces measuring approximately 2-3 mm in length (Figure 1A) and immediately immerse it in the fixative (the cut tissue will float on the fixative).

The tissue/fixative is placed in a desiccator, which is connected to a diaphragm vacuum pump. The tissue/fixative is vacuum degasified for 5 min at room temperature using the vacuum pump before releasing the vacuum very slowly. Pull and release the vacuum again until the tissue sinks.

Incubate overnight at 4 °C without vacuum.

Dissolve 5.0 g agar powder in 100 ml of PBS using a microwave oven. Place the molten agar medium on the hotplate/stirrer, which must be preheated to 60 °C.

Remove the fixative. Rinse the tissue three times with PBS. Pour the tissue into a paraffin dish.

Glue the agar block, in which pieces of cut leaf blade tissues were placed upright, onto the stage of a Leica VT1200S vibrating blade microtome with a drop of instant glue (Konishi Aron Alpha) (Figure 1D-E and Figure 2B).

Cut the agar-embedded tissue transversely, set upright in the agar block, with a thickness of 70 μm with the vibroslicer (Figure 1F-H and Figure 2C). Collect the transverse sections in PBS by tweezers.

Figure 2. Schematic drawing of section preparation. Rice leaf blade tissues are embedded in agar (A), and the agar block is glued on the stage of a Leica VT1200S vibrating blade microtome (B). The agar-embedded tissue is cut parallel to the surface of the stage (shown as dotted lines in C).

Transfer the sections and incubate them in sequential dehydration treatments with 0.5 ml of 70%, 80%, 90%, and 100% ethanol for 30 min in each solution without shaking.

The sections are then transferred and incubated for 30 min in each step in increasing concentrations of benzene in ethanol, ranging from 10% up to 100% with 10% stepwise increases, until the solvent has been replaced with benzene.

The benzene-equilibrated sections are stained with 0.1% crystal violet lactone solution for 10 min to visualize the silicified cells.

Place the stained sections on a microscope slide and cover it gently with a cover slip. Seal the cover slip with nail polish.

The sections are observed using an optical microscope and images are recorded using a CCD camera (Figure 2).

This protocol has been adapted or modified from a previous study by Isa et al. (2010). This study was supported by the Japan Society for Promotion of Science (JSPS) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan [a Grant-in-Aid for Scientific Research on Innovative Areas “Plant Cell Wall” (No. 24114001, 24114005) to K.N. and a Grant-in-Aid for Scientific Research (C) (25440124) to R.Y.].

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